Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

Emily A. Rex; Dahee Seo; Don B. Gammon

doi:10.3791/58244

Infezioni da arbovirus come strumenti di Screening per l'identificazione di fattori virali immuno...

JoVE Journal
Immunology and Infection

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JoVE Journal Immunology and Infection

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

Infezioni da arbovirus come strumenti di Screening per l'identificazione di fattori virali immunomodulatori e Host antivirale

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06:02 min

September 13, 2018

DOI: 10.3791/58244-v

Emily A. Rex¹, Dahee Seo¹, Don B. Gammon¹

¹Department of Microbiology,University of Texas Southwestern Medical Center

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Overview

This article presents protocols to identify virus-encoded immunomodulators that promote arbovirus replication and eukaryotic host factors that restrict it. The methods described utilize fluorescence and luminescence to provide quantitative readouts of arbovirus replication in simplified assays.

Key Study Components

Area of Science

Viral immunology
Arbovirus replication
Host-pathogen interactions

Background

Understanding host factors that restrict arbovirus replication is crucial.
Identifying virus-encoded immune evasion proteins can reveal mechanisms of viral persistence.
Lepidopteran insect cells provide a unique model for studying arbovirus replication.
Minimal background replication in these cells aids in detecting conditions for successful viral replication.

Purpose of Study

To elucidate host factors that limit arbovirus replication.
To identify viral proteins that counteract host restriction mechanisms.
To develop a reliable assay for studying arbovirus behavior in a controlled environment.

Methods Used

Maintenance of LD652 cells in tissue culture dishes.
Passaging cells at 80% confluency.
Using luminescence and fluorescence assays to measure viral replication.
Transferring diluted cell cultures to 24-well plates for experimentation.

Main Results

Successful identification of host factors that restrict arbovirus replication.
Characterization of virus-encoded proteins that facilitate replication.
Demonstration of effective assay conditions for studying arbovirus behavior.
Insights into the dynamics of host-pathogen interactions in lepidopteran cells.

Conclusions

The methods developed provide a framework for future studies in viral immunology.
Understanding the interplay between host factors and viral proteins can inform therapeutic strategies.
This research enhances our knowledge of arbovirus replication mechanisms.

Frequently Asked Questions

What are arboviruses?

Arboviruses are viruses transmitted by arthropods, such as mosquitoes and ticks, that can cause diseases in humans and animals.

Why use lepidopteran cells for this study?

Lepidopteran cells have minimal background replication of arboviruses, making them ideal for studying viral replication conditions.

What is the significance of identifying host factors?

Identifying host factors helps understand how viruses evade immune responses and can lead to the development of targeted therapies.

How do fluorescence and luminescence assays work?

These assays measure the light emitted by fluorescent or luminescent markers, providing quantitative data on viral replication.

What are the implications of this research?

The findings can inform future research on viral pathogenesis and the development of antiviral strategies.

Qui, presentiamo i protocolli per identificare gli immunomodulatori 1) virus-messo che promuovono la replica da arbovirus e fattori ospite 2) eucariotica che limitano la replica da arbovirus. Questi metodi basati su fluorescenza e luminescenza permettono ai ricercatori di ottenere rapidamente letture quantitative della replica da arbovirus nelle analisi semplicistiche con bassi rapporti segnale-rumore.

Questo metodo può aiutare a rispondere a domande chiave nel campo dell'immunologia virale, come ad esempio, quali fattori dell'ospite limitano la replicazione dell'arbovirus e, a loro volta, quali proteine di evasione immunitaria codificate dal virus superano questi meccanismi di restrizione dell'ospite. Il vantaggio principale di questa tecnica è che consente di utilizzare la luminescenza semplicistica e i saggi spaziali fluorescenti per definire le condizioni cellulari che consentono agli arbovirus di replicarsi in un'infezione altrimenti abortita nelle cellule di insetti lepidotteri. Poiché la replicazione di fondo degli arbovirus nelle cellule di lepidotteri è minima, diventa relativamente semplice rilevare le condizioni che consentono la replicazione degli arbovirus.

Per iniziare, mantenere le cellule LD652 in piastre trattate con coltura tissutale di 10 centimetri e far passare le cellule quando raggiungono l'80% di confluenza. Pipettare ripetutamente il terreno sul monostrato cellulare per rimuovere le cellule, quindi diluire il campione nel terreno di crescita fino a una densità approssimativa di 100 mila cellule per millilitro. Successivamente, trasferire un millilitro della coltura diluita in ciascun pozzetto di una piastra a 24 pozzetti.

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